A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells

BACKGROUND: Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy with bispecific diabodies could be a promising novel treatment option for prostate cancer. METHODS: A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv V_HCD3-V_LPSMA and V_HPSMA-V_LCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model. RESULTS: By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor xenografts with the diabody and PBL efficiently inhibited tumor growth. CONCLUSIONS: The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease

РАДИКАЛЬНАЯ ПРОСТАТЭКТОМИЯ И РОБОТИЗИРОВАННАЯ РАДИОХИРУРГИЯ: МЕТОДЫ ВЫБОРА ЛЕЧЕНИЯ ПАЦИЕНТОВ С ЛОКАЛИЗОВАННЫМ РАКОМ ПРЕДСТАТЕЛЬНОЙ ЖЕЛЕЗЫ

Purpose: to compare immediate, short-term and long-term treatment outcomes after radical prostatectomy and robotic radiosurgery in patients with localized prostate cancer. Material and methods. The study included 2 groups of patients. Group I patients (n=80) underwent radical surgery (nerve-sparing prostatectomy). Group II patients (n=102) underwent KiberKnife stereotactic robotic radiotherapy. Results. Immediate treatment outcomes after both radical prostatectomy and robotic radiosurgery did not demonstrate increased number of postoperative complications and severe radiation-induced injuries. The PSA level decreased and reached a nadir PSA level immediately after radical prostatectomy. After stereotactic radiation therapy, the PSA level decreased gradually every three months, reaching a nadir within a year. In the group of patients receiving CyberKnife treatment, changes in the prostate volume affected the quality of urination, which was confirmed by the assessment of the volume of residual urine. When comparing the quality of urination in treatment groups using the IPSS scale, the values were comparable. No differences in the 1-year disease-free survival rates between groups were found. However, 60 months later, disease free survival rate was higher by 8.2 % in patients treated with radiosurgery than in patients who underwent radical prostatectomy (p<0.005). No differences in the 1-year cancer-specific survival rates between the groups were found. In patients, who underwent radiosurgery, the 5-year cancer-specific survival rate was 96.3 % (3.7 % higher than that observed in patients, who underwent radical surgery, p<0.005).Conclusion. High 5-year survival rates in patients with localized prostate cancer indicate that both radical prostatectomy and KiberKnife stereotactic robotic radiosurgery are safe and effective treatment options.Цель исследования – сравнить показатели непосредственных, ближайших и отдаленных результатов лечения после радикальной простатэктомии и роботизированной радиохирургии у пациентов с локализованным раком предстательной железы. Материал и методы. В исследование включены 2 группы пациентов: первая группа – 80 больных, получивших хирургическое лечение (радикальная позадилонная нервосберегающая простатэктомия), вторая группа – 102 пациента, которым проведена роботизированная стереотаксическая лучевая терапия на аппарате «КиберНож». Результаты. Как после хирургического лечения, так и после радиохирургии не наблюдается значительного количества послеоперационных осложнений и выраженных лучевых реакций. После радикальной простатэктомии уровень ПСА уменьшается, достигая надира сразу после проведенного лечения. После стереотаксической лучевой терапии уровень ПСА уменьшается постепенно, через каждые 3 мес достигая надира в течение года. В группе пациентов после лечения на аппарате «КиберНож» динамика изменения объема предстательной железы отражается на качестве мочеиспускания пациентов, что подтверждается оценкой показателей объема остаточной мочи, при сравнении качества мочеиспускания в группах по шкале IPSS значения сопоставимы. При анализе отдаленных результатов лечения показатели одногодичной безрецидивной выживаемости в группах сопоставимы, однако через 60 мес безрецидивная выживаемость в группе пациентов после радиохирургии больше на 8,2 % (p<0,005). Показатели одногодичной онкоспецифической выживаемости сопоставимы. Показатели 5-летней онкоспецифической выживаемости у больных после радиохирургии составили 96,3 %, что на 3,7 % больше, чем после радикального хирургического лечения (p<0,005). Заключение. Предложенные варианты лечения пациентов с локализованным раком предстательной железы являются безопасными, а высокие показатели онкоспецифической 5-летней выживаемости свидетельствуют об эффективности как радикальной позадилонной нервосберегающей простатэктомии, так и роботизированной стереотаксической лучевой терапии на аппарате «КиберНож».

Brachytherapy 1-125 in the treatment of patients with prostate cancer

Bricherasio. Analyzed the immediate and remote results of treatment of patients with prostate cancer who received combined treatment - neoadjuvant hormone therapy in combination with brachytherapy 1-125. Immediate results of treatment were estimated by the level of total PSA, prostate volume, residual urine, change in the international scale IPSS. The dynamics of the above indicators comparable in both groups, which is reflected in the regression of the primary lesion, as well as the quality of life of patients. One-year disease-free survival in the group of patients who received comprehensive treatment amounts to 71.4 %, in the group of patients who underwent only interstitial radiation therapy to 92.3 %; Overall survival, as well as ecospecrfier survival in both groups is 100%.Проанализированы ближайшие и отдаленные результаты лечения больных раком предстательной железы, получивших комплексное лечение - неоадьювантную гормонотерапию в сочетании брахитерапией 1-125. Ближайших результатов лечения оценены по уровню общего ПСА, объему предстательной железы, количеству остаточной мочи, изменение по международной шкале IPSS. Динамика вышеперечисленных показателей сопоставима в обеих группах, что отражается на регрессии первичного очага, а также качестве жизни пациентов. Одногодичная безрецидивная выживаемость в группе пациентов получивших комплексное лечение составляет 71,4 %, в группе пациентов которым проведена только внутритканевая лучевая терапия 92,3 %; Общая выживаемость, а также онкоспецифическая выживаемость в обеих группах составляет 100%

Single-Batch Production of Recombinant Human Polyclonal Antibodies

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the SympressTM I technology. The SympressTM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the SympressTM I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the SympressTM II technology. Here we describe proof-of-principle data demonstrating the feasibility of the SympressTM II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase

РОБОТИЗИРОВАННАЯ РАДИОХИРУРГИЯ КАК ВАРИАНТ ЛЕЧЕНИЯ ПАЦИЕНТОВ С ЛОКАЛИЗОВАННЫМ РАКОМ ПРЕДСТАТЕЛЬНОЙ ЖЕЛЕЗЫ

The article analyzed the immediate and long-term results of treatment of patients with prostate cancer receiving treatment method – robotic radiosurgery. The data of the immediate results of treatment are evaluated on the following criteria: level of total PSA, prostate volume, residual urine, changes on the international scale IPSS. The dynamics of the above indicators shows the regression of the primary focus, and the absence of pronounced radiation reactions affect the quality of life of patients. Three-year disease-free survival in patients receiving radiation therapy is 88.8 %. Overall survival, cancer-specific survival as well as 100 %. Проанализированы ближайшие и отдаленные результаты лечения 48 больных раком предстательной железы II стадии, получивших лечение методом роботизированной радиохирургии. Ближайшие результаты лечения оценивались по динамике следующих показателей: уровеню общего ПСА, объему предстательной железы, количеству остаточной мочи, степени нарушений мочеиспускания по международной шкале IPSS. Отмечены выраженная регрессия первичного очага и отсутствие выраженных лучевых реакций, что позитивно отражается на качестве жизни пациентов. Трехлетняя безрецидивная выживаемость составляет 88,8 %, общая и онкоспецифическая выживаемость – 100 %.

Эффективность применения лучевых методов лечения пациентов с локализованным раком предстательной железы в условиях одного стационара

Objectives. Analyzed short-and long-term results of radiation therapies (brachytherapy 125I, conformal radiation therapy, stereotactic robotic radiotherapy) of patients with prostate cancer.Materials and methods. In each treatment the patients were divided into 2 groups received the combined treatment in combination with neoadjuvant hormone therapy without its implementation. The immediate results of treatment were assessed according to the level of total prostatic specific antigen, prostate volume, residual urine, change according to the International Prostatic Symptom Score (IPSS).Results. The dynamics of these indicators comparable in both groups, which is reflected in the regression of the primary tumor, as well as the quality of life of patients. Five-year overall survival and oncospecific survival in the group of patients after stereotactic radiotherapy is 100 %, the remaining groups – of more than 90–95 %.Conclusion. The obtained results testify to the effectiveness of the proposed methods of treatment of patients with localized prostate cancer. Цель работы – оценка показателей ближайших и отдаленных результатов применения лучевых методов лечения (брахитерапии источниками йода-125, конформной лучевой терапии, стереотаксической роботизированной радиотерапии) больных раком предстательной железы.Материалы и методы. В каждом представленном методе терапии пациенты были разделены на 2 подгруппы: больные 1-й подгруппы получали комбинированное лечение в сочетании с неоадъювантной гормонотерапией, пациенты 2-й подгруппы – лучевой метод лечения в монорежиме. Ближайшие результаты терапии оценивали по уровню общего простатического специфического антигена, объему предстательной железы, количеству остаточной мочи и изменению по Международной шкале оценки простатических симптомов (International Prostatic Symptom Score, IPSS).Результаты. Динамика этих показателей была сопоставима в обеих подгруппах, что отражается на регрессии первичного очага, а также на качестве жизни пациентов. Онкоспецифическая и 5-летняя общая выживаемость в группе пациентов после проведения стереотаксической радиотерапии составила 100 %, в остальных группах – более 90–95 %.Заключение. Полученные результаты свидетельствуют об эффективности предложенных методов лечения пациентов с локализованным раком предстательной железы.

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices

[EN] A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against (RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl-1,1,2,2-tetrafluoroethyl ether (tetraconazole), 2-(4-triazolyl)benzimidazole (thiabendazole), and (RS)-1-(beta-allyloxy-2,4-dichlorophenylethyl)imidazole (imazalil) were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices.This work was funded by Ministerio de Educacion y Ciencia (MEC, Spain, Project AGL2002-03266). E. P. was the recipient of a doctoral fellowship from Conselleria d'Educacio (Generalitat Valenciana, Spain).Moreno Tamarit, MJ.; Plana Andani, E.; Manclus Ciscar, JJ.; Montoya Baides, Á. (2014). Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices. Food Analytical Methods. 7(2):481-489. https://doi.org/10.1007/s12161-013-9655-zS48148972Abad A, Manclús JJ, Moreno M, Montoya A (2001) J AOAC Int 84:1–6Alcocer MJC, Doyen C, Lee HA, Morgan MRA (2000) J Agric Food Chem 48:4053–4059Brichta J, Vesela H, Franek M (2003) Vet Med 48:237–247Brichta J, Hnilova M, Viskovic T (2005) Vet Med 50:231–252Charlton K, Harris WJ, Potter AJ (2001) Biosens Bioelec 16:639–646EU Pesticide Database (2013) Pesticide EU-MRLs. http://ec.europa.eu/sanco_pesticides/public/index.cfm . Accessed Jan 2013Ferrer C, Martínez-Bueno MJ, Lozano A, Fernández-Alba AR (2011) Talanta 83:1552–1561Garret SD, Appleford DJA, Wyatt GM, Lee HA, Morgan MRA (1997) J Agric Food Chem 45:4183–4189Graham BM, Porter AJ, Harris WJ (1995) J Chem Technol Biotech 63:279–289Hiemstra M, de Kok A (2007) J Chromatog A 1154:3–25Kipriyanov SM, Moldenhauer G, Little M (1997) J Immunol Meth 200:69–77Kramer K, Hock B (2007) Recombinant antibodies for agrochemicals: Evolutionary optimization. In: Kennedy IR, Solomon KR, Gee SJ, Crossan AN, Wang S, Sánchez-Bayo F (eds) Rational environmental management of agrochemicals: Risk assessment, monitoring, and remedial action. ACS Symposium Series, vol. 966, pp 155−170Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A (1997) J Immunol Meth 201:35–55Leong SSJ, Chen WN (2008) Chem Engin Sci 63:1401–1414Li T, Zhang Q, Liu Y, Chen D, Hu B, Blake DA, Liu F (2006) J Agric Food Chem 54:9085–9091Manclús JJ, Moreno M, Plana E, Montoya A (2008) J Agric Food Chem 56:8790–8800Markus V, Janne L, Urpo L (2011) Trends Anal Chem 30:219–226Mersmann M, Schmidt A, Tesar M, Schöneberg A, Welschof M, Kipriyanov S, Terness P, Little M, Pfizenmaier K, Moosmayer D (1998) J Immunol Meth 220:51–58Moreno M, Plana E, Montoya A, Caputo P, Manclús JJ (2007) Food Addit Contam 24:704–712Morozova VS, Levashova AI, Eremin SA (2005) J Anal Chem 60:202–217Nishi K, Imajuku Y, Nakata M, Ohde K, Miyake S, Morimune K, Kawata M, Ohkawa H (2003) J Pest Sci 28:301–309Nishi K, Ishiuchi M, Morimune K, Ohkawa H (2005) J Agric Food Chem 53:5096–5104Scholthof KB, Whang G, Karu AE (1997) J Agric Food Chem 45:1509–1517Sheedy C, MacKenzie CR, Hall JC (2007) Biotech Adv 25:25333–25352Tout NL, Yau KYF, Trevors JT, Lee H, Hall JC (2001) J Agric Food Chem 49:3628–3637Webb SR, Lee H, Hall JC (1997) J Agric Food Chem 45:535–541Yau KYF, Tout NL, Trevors JT, Lee H, Hall JC (1998) J Agric Food Chem 46:4457–4463Yoshioka N, Akiyama Y, Matsuoka T, Mitsuhashi T (2010) Food Control 21:212–21

Antibody Engineering Using Phage Display with a Coiled-Coil Heterodimeric Fv Antibody Fragment

A Fab-like antibody binding unit, ccFv, in which a pair of heterodimeric coiled-coil domains was fused to VH and VL for Fv stabilization, was constructed for an anti-VEGF antibody. The anti-VEGF ccFv showed the same binding affinity as scFv but significantly improved stability and phage display level. Furthermore, phage display libraries in the ccFv format were constructed for humanization and affinity maturation of the anti-VEGF antibody. A panel of VH frameworks and VH-CDR3 variants, with a significant improvement in affinity and expressibility in both E. coli and yeast systems, was isolated from the ccFv phage libraries. These results demonstrate the potential application of the ccFv antibody format in antibody engineering